In recent decades, molecular biology studies have focused primarily on genes, the transcription of those genes into RNA, and the translation of the RNA into protein. There has been a more limited analysis of the regulatory mechanisms associated with gene control. Gene regulation, for example, at what stage of development of the individual a gene is activated or inhibited, and the tissue specific nature of this regulation is less well understood. However, such regulation can be with the extent and nature of methylation of the gene or genome. Specific cell types can be correlated with specific methylation patterns, as has been shown for a number of cases (Adorjan et al. (2002) Tumour class prediction and discovery by microarray-based DNA methylation analysis. Nucleic Acids Res. 30 (5) e21).
In higher order eukaryotes, DNA is methylated nearly exclusively at cytosines located 5′ to guanine in the CpG dinucleotide. This modification has important regulatory effects on gene expression, especially when involving CpG rich areas, known as CpG islands located in the promoter regions of many genes. While almost all gene-associated islands are protected from methylation on autosomal chromosomes, extensive methylation of CpG islands has been associated with transcriptional inactivation of selected imprinted genes and genes on the inactive X-chromosome of females.
Cytosine modification, in form of methylation, contains significant information. The identification of 5-methylcytosine in a DNA sequence, as opposed to unmethylated cytosine; that is, the methylation status, is of great importance and warrants further study. However, because 5-methylcytosine behaves like cytosine in terms of hybridization preference (a property relied on for sequence analysis), its positions/status can not be identified by a normal sequencing reaction. Furthermore, in any amplification, such as a PCR amplification, this relevant epigenetic information, methylated cytosine or unmethylated cytosine, will be lost completely.
Several methods are known in the art that relate to this problem. Usually genomic DNA is treated with a chemical or enzyme leading to a conversion of the cytosine bases, which consequently allows subsequent base differentiation. The most common methods are: a) the use of methylation sensitive restriction enzymes capable of differentiating between methylated and unmethylated DNA; and b) the treatment with a bisulfite reagent. The use of said enzymes is limited due to the selectivity of the restriction enzyme towards a specific recognition sequence.
Therefore, the specific reaction of bisulfite with cytosine, which, upon subsequent alkaline hydrolysis is converted to uracil (whereas 5-methylcytosine remains unmodified under these conditions) (Shapiro et al. (1970) Nature 227: 1047) is currently the most frequently used method for analyzing DNA for 5-methylcytosine. Uracil corresponds to thymine in its base pairing behaviour; that is, it hybridizes to adenine; whereas 5-methylcytosine does not change its chemical properties under this treatment, and therefore still has the base pairing behavior of a cytosine (hybridizing with guanine). Consequently, the original DNA is converted in such a manner that 5-methylcytosine, which originally could not be distinguished from cytosine by its hybridization behavior, can now be detected as the only remaining cytosine using standard molecular biological techniques, for example, amplification and hybridization or sequencing. All of these techniques are based on base pairing, which can now thereby be more fully exploited. Comparing the sequences of the DNA with and without bisulfite treatment allows an easy identification of those cytosines that have been unmethylated. An overview of further known methods for detecting 5-methylcytosine may be gathered from the following review article: Fraga & Esteller, Biotechniques 33:632, 634, 636-49, 2002.
Again, because the use of methylation-specific enzymes is restricted to certain sequences (comprising restriction sites), most typical methods are based on a bisulfite treatment that is conducted before a detection or amplifying step (for review: DE 100 29 915, A1 p.2, lines 35-46 or the according translated U.S. application Ser. No. 10/311,661, see also WO 2004/067545 ). The term ‘bisulfite treatment’ in this context is meant to comprise treatment with a bisulfite, a disulfite or a hydrogensulfite solution. As known to a person or ordinary skill in the art (and as used herein), the term “bisulfite” is used interchangeably for “hydrogensulfite”.
Several bisulfite-based protocols are known in the art. However, all of the described protocols, comprise the following steps: the genomic DNA is isolated, denatured, converted several hours by a concentrated bisulfite solution and finally desulfonated and desalted (see, e.g., Frommer et al.: A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci USA. 89:1827-31, 1992).
Recent technical improvements of bisulfite methods. The art-recognized agarose bead method incorporates the DNA to be investigated in an agarose matrix, through which diffusion and renaturation of the DNA is prevented (bisulfite reacts only on single-stranded DNA) and all precipitation and purification steps are replaced by rapid dialysis (Olek A. et al. A modified and improved method for bisulphite based cytosine methylation analysis, Nucl. Acids Res. 24, 5064-5066, 1996).
Patent application WO 01/98528 (20040152080) describes a bisulfite conversion in which the DNA sample is incubated with a bisulfite solution of a concentration range between 0.1 mol/l to 6 mol/l in the presence of a denaturing reagent and/or solvent and at least one scavenger. In said patent application, several suitable denaturing reagents and scavengers are described. Desulfonation of the deaminated nucleic acid is afforded by incubation of the solution under alkaline conditions.
Patent application WO 03/038121 (US 20040115663) discloses a method in which the DNA to be analysed is bound to a solid surface during the bisulfite treatment. Consequently, purification and washing steps are facilitated.
Patent application WO 04/067545 discloses a method in which the DNA sample is denatured by heat and incubated with a bisulfite solution of a concentration range between 3 mol/l to 6.25 mol/l. Thereby the pH value is between 5.0 and 6.0 and the nucleic acid is deaminated. Deaminated nucleic acids are desulfonated by incubation of the solution under alkaline conditions.
The art-recognized understanding that a ‘bisulfite conversion’ typically comprises a desulfonation step is illustrated in WO 04/067545:                “According to the invention the term a “bisulfite reaction”, “bisulfite treatment” or “bisulfite method” shall mean a reaction for the conversion of a cytosine base, preferably cytosine bases, in a nucleic acid to an uracil base, preferably uracil bases, in the presence of bisulfite ions whereby preferably a 5-methyl-cytosine base, preferably 5-methyl-cytosine bases, is not significantly converted. This reaction for the detection of methylated cytosines is described in detail by Frommer et al., supra and Grigg and Clark (Grigg, G. and Clark, S., Bioessays 16:431-436, 1994). The bisulfite reaction contains a deamination step and a desulfonation step, which can be conducted separately or simultaneously (see FIG. 1; Grigg and Clark, supra). The statement that 5-methyl-cytosine bases are not significantly converted shall only take the fact into account that it cannot be excluded that a small percentage of 5-methyl-cytosine bases is converted to uracil although it is intended to convert only and exclusively the (non-methylated) cytosine bases (Frommer et al., supra). The expert skilled in the art knows how to perform the bisulfite reaction, e.g. by referring to Frommer et al., supra or Grigg and Clark, supra who disclose the principal parameters of the bisulfite reaction.”        
Moreover, WO 04/067545 describes the general state of the art with regard to the different protocols:                “From Grunau et al., supra, it is known to the expert in the field what variations of the bisulfite method are possible. In summary, in the deamination step a buffer containing bisulfite ions, optionally chaotropic agents and optionally further reagents as an alcohol or stabilizers as hydroquinone are employed and the pH is in the acidic range. The concentration of bisulfite is between 0.1 and 6 M bisulfite, preferably between 1 M and 5.5 M, the concentration of the chaotropic agent is between 1 and 8 M, whereby preferably guanidinium salts are employed, the pH is in the acidic range, preferably between 4.5 and 6.5, the temperature is between 0° C. and 90° C., preferably between room temperature (25° C.) and 90° C., and the reaction time is between 30 min and 24 hours or 48 hours or even longer, but preferably between 1 hour and 24 hours. The desulfonation step is performed by adding an alkaline solution or buffer as e.g. a solution only containing a hydroxide, e.g. sodium hydroxide, or a solution containing ethanol, sodium chloride and sodium hydroxide (e.g., 38% EtOH, 100 mM NaCl, 200 mM NaOH) and incubating at room temperature or elevated temperatures for several min, preferably between 5 min and 60 min.”        
Desulfonation is, therefore, an inherent feature of all of these methods, and in any case a desulfonation takes place before the nucleic acids are used as templates for amplification reactions, in order to provide an ideal template for the polymerase utilized in subsequent reactions.
Patent application WO 05/038051 describes improvements for the conversion of unmethylated cytosine to uracil by treatment with a bisulfite reagent. According to this method the reaction is carried out in the presence of 10-35% by volume, preferentially in the presence of 20-30% by volume of dioxane, one of its derivatives or a similar aliphatic cyclic ether. The bisulfite reaction can also be carried out in the presence of a n-alkylene glycol compound, particularly in the presence of their dialkyl ethers, and especially in the presence of diethylene glycol dimethyl ether (DME). These compounds can be present in a concentration of 1-35% by volume, preferentially of 5-25% by volume. The bisulfite conversion is conducted at a temperature in the range of 0-80° C. and that the reaction temperature is increased for 2 to 5 times to a range of 85-100° C. briefly during the course of the conversion (thermospike). It is further preferred that the temperature increases to 85-100° C., in particular to 90-98° C. during the temperature increase of brief duration.
Subsequent to a bisulfite treatment, usually short, specific fragments of a known gene are amplified and either completely sequenced (Olek A, Walter J, The pre-implantation ontogeny of the H19 methylation imprint. Nat Genet. 3:275-6, 1997) or individual cytosine positions are detected by a primer extension reaction (Gonzalgo M L and Jones P A., Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE). Nucleic Acids Res. 25:2529-31, 1997; WO 95/00669) or by enzymatic digestion (Xiong Z, Laird P W., COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res. 25: 2535-4, 1997).
Another technique to detect hypermethylation is the so-called methylation specific PCR (MSP) (Herman J G, Graff J R, Myohanen S, Nelkin B D and Baylin S B., Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A. 93: 9821-6, 1996). The technique is based on the use of primers that differentiate between a methylated and a non-methylated sequence if applied after bisulfite treatment of said DNA sequence. The primer either contains a guanine at the position corresponding to the cytosine in which case it will after bisulfite treatment only bind if the position was methylated. Or the primer contains an adenine at the corresponding cytosine position and therefore only binds to said DNA sequence after bisulfite treatment if the cytosine was unmethylated and has hence been altered by the bisulfite treatment so that it hybridizes to adenine. With the use of these primers, amplicons can be produced specifically depending on the methylation status of a certain cytosine and will as such indicate its methylation state.
Another technique is the detection of methylation via a labeled probe, such as used in the so called Taqman™ PCR, also known as MethyLight™ (U.S. Pat. No. 6,331,393). With this technique it became feasible to determine the methylation state of single or of several positions directly during PCR, without having to analyze the PCR products in an additional step.
Additionally, detection by hybridization has also been described (Olek et al., WO 99/28498).
The treatment with bisulfite (or similar chemical agents or enzymes) with the effect of altering the base pairing behaviour of one type of cytosine specifically, either the methylated or the unmethylated, thereby introducing different hybridisation properties, makes the treated DNA more applicable to the conventional methods of molecular biology, especially the polymerase based amplification methods, such as the PCR.
Base excision repair. Base excision repair occurs in vivo to repair DNA base damage involving relatively minor disturbances in the helical DNA structure, such as deaminated, oxidized, alkylated or absent bases. Numerous DNA glycosylases are known in the art, and function in vivo during base excision repair to release damaged or modified bases by cleavage of the glycosidic bond that links such bases to the sugar phosphate backbone of DNA (Memisoglu, Samson, Mutation Res., 451:39-51, 2000). All DNA glycosylases cleave glycosidic bonds but differ in their base substrate specificity and in their reaction mechanisms.
Carry-over contamination of amplification reactions (e.g., PCR); inadequacy of the prior art. One widely recognized application of such glycosylases is decontamination in PCR applications. In any such PCR amplification, 2 to the 30 (230) or more copies of a single template are generated. This very large amount of DNA produced helps in the subsequent analysis, like in DNA sequencing according to the Sanger method, but it can also become a problem when this amount of DNA is handled in an analytical laboratory. Even very small reaction volumes, when inadvertently not kept in a closed vial, can lead to contamination of the whole work environment with a huge number of DNA copies. These DNA copies may be templates for a subsequent amplification experiment performed, and the DNA analysed subsequently may not be the actual sample DNA, but contaminating DNA from a previous experiment. This may also lead to positive negative controls that should not contain any DNA and therefore no amplification should be observed.
In practice, this problem can be so persistent that whole laboratories may move to a new location, because contamination of the work environment makes it impossible to still carry out meaningful PCR based experiments. In a clinical laboratory, however, the concern is also that contaminating DNA may cause false results when performing molecular diagnostics. This would mean that actually contaminating DNA that stems from a previous patient is analyzed, instead of the actual sample to be investigated.
Therefore, measures have been implemented to avoid contamination. This involves, for example, a PCR amplification and detection in one tube in a real time PCR experiment. In this case, it is not required that a PCR tube be opened. After use, the tube will be kept closed and discarded and therefore the danger of contamination leading to false results is greatly reduced.
In addition, molecular means exist that reduce the risk of contamination. In a polymerase chain reaction, the enzyme uracil-DNA-glycosylase (UNG) reduces the potential for false positive reactions due to amplicon carryover (see e.g. U.S. Pat. No. 5,035,996 or Thornton C G, Hartley J L, Rashtchian A., Utilizing uracil DNA glycosylase to control carryover contamination in PCR: characterization of residual UDG activity following thermal cycling. Biotechniques, 13:1804, 1992). The principle of this contamination protection method is that in any amplification instead of dTTP dUTP is provided and incorporated and the resulting amplicon can be distinguished from its template and any future sample DNA by uracil being present instead of thymine. Prior to any subsequent amplification, uracil DNA-glycosylase (UNG) is used to cleave these bases from any contaminating DNA, and therefore only the legitimate template remains intact and can be amplified. This method is considered the standard method of choice in the art and is widely used in DNA based diagnostics. The following is a citation from a publication that summarizes the use of UNG (Longo M C, Berninger M S, Hartley J L., Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene., 93:125-8, 1990):                “Polymerase chain reactions (PCRs) synthesize abundant amplification products. Contamination of new PCRs with trace amounts of these products, called carry-over contamination, yields false positive results. Carry-over contamination from some previous PCR can be a significant problem, due both to the abundance of PCR products, and to the ideal structure of the contaminant material for re-amplification. We report that carry-over contamination can be controlled by the following two steps: (i) incorporating dUTP in all PCR products (by substituting dUTP for dTTP, or by incorporating uracil during synthesis of the oligodeoxyribonucleotide primers; and (ii) treating all subsequent fully preassembled starting reactions with uracil DNA glycosylase (UNG), followed by thermal inactivation of UNG. UNG cleaves the uracil base from the phosphodiester backbone of uracil-containing DNA, but has no effect on natural (i.e., thymine-containing) DNA. The resulting apyrimidinic sites block replication by DNA polymerases, and are very labile to acid/base hydrolysis. Because UNG does not react with dUTP, and is also inactivated by heat denaturation prior to the actual PCR, carry-over contamination of PCRs can be controlled effectively if the contaminants contain uracils in place of thymines.”        
Another method for carry over protection in PCR has been described by Walder et al (Walder R Y, Hayes J R, Walder J A., Use of PCR primers containing a 3-terminal ribose residue to prevent cross-contamination of amplified sequences. Nucleic Acids Res., 21:4339-43, 1993). Walder et al describe that carry over protection can be achieved—however not very reproducibly—by using primers consisting of a 3′-end which is characterized as a ribo-cytidine. After primer extension the amplification product is cleaved specifically at the site of this ribonucleotide by an enzyme known as RNase A. That way the potentially contaminating amplificates are shortened at their ends and cannot serve a templates for said primers in the following amplification procedure. However, a significant disadvantage inherent to this method is the instability of the primer molecules, containing a ribonucleotide at the 3′-end.
All of the documents cited herein are hereby incorporated by reference in its entirety.
Substantial problem in the prior art. Because the existence of uracils is an inherent feature of bisulfite converted DNA and the necessary property relied upon for detecting methylation differences, the prior art method of choice for carry over protection based on uracil-DNA-glycosylase enzyme activity, as described above, cannot be applied. This limitation is very unfortunate, because a number of powerful assays for diagnosis are based on PCR performed on bisulfite converted DNA as a template. The difficulty of solving the problem for decontamination of bisulfite converted templates is considered a general one, that can not be solved by adaptation of the standard UNG method, as any bisulfite converted DNA will contain uracil as well. It has therefore commonly been argued that, in any uracil-DNA-glycosylase step, the template DNA would be destroyed along with any contaminating DNA.
Therefore, there is a pronounced need in the art for new methods for carry over prevention that have utility for routine performance of such assays. There is a pronounced need in the art to provide solutions to the problem of how to achieve a reliable carry over protection when analysing methylation of cytosine positions in DNA from patient samples.